An important goal of physical therapy, sports medicine and many complementary and alternative (CAM) therapies is the prevention of injuries through promotion and maintenance of optimal musculoskeletal function. A wide variety of treatments including manual and movement-based therapies are concerned with optimizing body alignment, posture, biomechanics and quality of movement. Many of these treatments involve stretching of soft tissues within or slightly beyond their usual range of motion, either manually or actively. Despite this widespread use, the effect of tissue stretch is poorly understood, with the majority of research so far being on muscle, with comparatively little on connective tissue. In our previous grant, we demonstrated a novel, dynamic response of connective tissue fibroblasts to tissue stretch. Static stretching of "loose" inter-fascial connective tissue causes extensive fibroblast cytoskeletal remodeling within 10 minutes, followed within 90 minutes by up-regulation of a large group of skeletal muscle- related genes. We also found that similar effects occur with acupuncture needle rotation due to winding and gathering of collagen around the rotating needle, essentially stretching the tissue "from the inside". A potentially important difference between acupuncture and tissue stretch, however, is that a "whorl" of collagen formed during needle rotation can persist after the needle has been withdrawn, thus causing a more prolonged stretching of tissue compared with simple stretch followed by release. In this project Renewal, we propose to expand our investigation of this active fibroblast response to tissue stretch, with the goals of characterizing its time course and dose-response characteristics (Aim 1), mechanotransduction mechanisms linking cytoskeletal and nuclear remodeling to intracellular signaling and gene expression (Aim 2) as well as downstream effects on fibroblast muscle-related protein expression and connective tissue tension regulation (Aim 3). We will use ex vivo and in vivo mouse models developed in our previous grant. Our results will be relevant to basic cell biology, connective tissue physiology, manual and movement based therapies as well as acupuncture. We believe that understanding this new phenomenon will lead to fundamental insights into the role played by mechanical stimulation in maintaining healthy connective tissue function. This expanded knowledge of normal physiology will potentially lead to discovering types of connective tissue dysfunction that are not currently recognized. An improved understanding of connective tissue physiology and pathophysiology will therefore provide a fundamentally broader and more solid foundation for understanding the therapeutic mechanisms of a wide range of conventional and alternative therapies and practices aimed at improving wellness, preventing injuries and treating musculoskeketal pain. PUBLIC HEALTH RELEVANCE. Chronic musculoskeletal pain resulting from acute or repetitive injuries is a major source of disability, work absenteeism and health case costs. In addition to treating pain, an important goal of physical therapy, sports medicine and many complementary and alternative (CAM) therapies is the prevention of injuries through promotion and maintenance of optimal musculoskeletal function. This project will investigate a novel response of connective tissue to mechanical stimulation, initially described in our previous grant, that will broaden the foundation for an expanded understanding of musculoskeletal wellness, prevention and treatment.